Kidney-targeted epoxyeicosatrienoic acid (eet) analogs

ABSTRACT

Described here are EET analogs conjugated to folate receptor ligands such as folic acid or folic acid analogs recognized by and selectively bound by folate receptors and other folate binding proteins and the use of such conjugated EET analogs for targeted delivery of therapeutic agents to folate-receptor bearing cell populations. More particularly, provided herein are EET analogs conjugated to folate receptor ligands and uses of such conjugated EET analogs as kidney targeted therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/647,603 filed on Mar. 3, 2020 which was the national stage filingunder 35 U.S.C. 371 of International Application No. PCT/US2018/051132,filed Sep. 14, 2018, which claims priority to U.S. Provisional PatentApplication No. 62/559,355, filed Sep. 15, 2017, each of which areincorporated herein by reference as if set forth in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

BACKGROUND

According to the National Kidney Foundation, 10% of the populationworldwide is affected by chronic kidney disease (CKD), and millions dieeach year because they do not have access to affordable or effectivetreatment. With the increasing prevalence of CKD and end-stage renaldisease (ESRD), the economic burden of CKD is a growing concern topatients, their caregivers, and payers. Significant health care costsare incurred to manage the clinical complexities of CKD and ESRDpatients, including costs associated with the detection and managementof CKD, ESRD treatment, and simultaneous management of comorbidconditions such as diabetes, hypertension, and congestive heart failure.Progressive organ damage associated with renal and cardiovasculardiseases is a major cause of morbidity and mortality. Hypertension anddiabetes are the two main diseases responsible for the increase in ESRDand the number of patients on dialysis. Despite increased awareness ofChronic Kidney Disease (CKD) as a significant worldwide medical problem,there remain few pharmacological therapies available to prevent or slowthe progression of CKD to End Stage Renal Disease (ESRD), much lesstherapies that result in a cure.

Drug-induced nephrotoxicity is also a problem for patients receivingplatinum-based chemotherapy agents for treatment of a variety ofmalignancies. For example, cisplatin is an effective chemotherapeuticagent, but several adverse side effects are associated with the use ofcisplatin in clinical practice. The most common adverse effect forpatients receiving cisplatin is severe nephrotoxicity which occurs in25-40% of cisplatin treated patients. It is widely acknowledged thatthere is a need to develop agent that could protect the kidney from theadverse effect of cisplatin during its clinical use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a chronic study protocol.

FIG. 2 demonstrates that a folate-conjugated form of an EET analogtargets to the kidney.

FIG. 3 demonstrates decreased cisplatin-induced renal injury using afolate conjugated form of an EET analog.

FIG. 4 illustrates an embodiment of a folate-conjugated EET analog.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

Described here and in the following pages are kidney targetedepoxyeicosatrienoic acid (EET) analogs and methods of using such EETanalogs for the treatment of renal and cardiovascular diseases. Moreparticularly, provided herein are EET analogs conjugated to folatereceptor ligands such as folic acid or folic acid analogs recognized byand selectively bound by folate receptors and other folate bindingproteins and uses of such conjugated EET analogs as kidney targetedtherapeutics. In general, these compounds and methods are based at leastin part on the inventor's development of EET agonists and analogs havingpromising therapeutic potential for cardiovascular and kidney diseases.The inventors previously synthesized a series of analogs of 14,15-EET,defined the basic pharmacophore and stabilized it from metabolicinactivation. A subset of these analogs, named EET-A, EET-B and EET-C22,are orally active with good pharmacokinetic properties. See, e.g.,PCT/US2012/032090 and U.S. Pat. Nos. 9,422,318 and 9,127,027, each ofwhich is incorporated herein by reference as if set forth in itsentirety.

In a first aspect, provided herein is a kidney-targeted analog ofepoxyeicosatrienoic acid (EET) comprising an EET analog conjugated tofolic acid (also known as folate) receptor ligand such as folic acid,methotrexate, or a folate analog that binds to the folate receptor.Without being bound to any particular theory or mechanism of action, itis believed that linkage of a folate receptor ligand to an EET analogtargets the folate-linked compound to cells expressing high levels offolate receptors (FR) such as proximal tubular cells of the kidney.Suitable folate receptor ligands include folic acid, methotrexate, andfolate analogs that bind to the folate receptor. The folate receptorligand may be directly conjugated to the EET analog, or alternatively,the folate receptor ligand may be indirectly conjugated to the EETanalog by a linker.

In some cases, the EET analog used to form the conjugated compound isselected from the group consisting of

In certain embodiments, the compound of the invention having thefollowing formula and further exhibits binding affinity for a folatereceptor:

Linkers and linking chemistry appropriate for folate-conjugation of EETanalogs for synthesis of kidney-targeted EET analogs include, withoutlimitation, Polyethylene glycol (PEG) linkers, PEG-diamine linkers. Forexample, in certain embodiments, the EET analog is conjugated to afolate receptor ligand via a PEG diamine linker. In other cases, thelinker comprises ethylenediamine (see, for example, FIG. 4),diisocyanate, diisothiocynate, carbodiimide, bis(hydroxysuccinimide)ester, maleimide-hydroxysuccinimide ester, glutaraldehyde, or acombination thereof.

The linker may create either a permanent or a semipermanent (i.e.,labile) linkage. The inclusion of a semipermanent linkage is especiallyadvantageous for applications in which cellular uptake of the drug isdesired.

Synthesis of kidney targeted EET analogs may be accomplished accordingto the following illustrative synthesis protocols. Synthesis of otherfolic acid analogs may be accomplished by methods known to the skilledartisan. In addition, the optional incorporation of a linker may also beaccomplished by methods known to the skilled artisan.

Synthesis of Folate-Conjugates

Synthesis of EET-F01:

Experimental:

Diisopropylethylamine (DIPEA, 1.18 mL, 6.78 mmol, 3.00 equiv) was addedto a mixture of N-hydroxysuccinimide (NHS) folate ester¹ (1) (11.44 g,2.26 mmol) and 2-[2-(2-aminoethoxy)ethoxy]-N-tritylethan-1-amine² (2)(1.06 g, 2.71 mmol, 1.20 equiv) in anhydrous DMSO (20 mL). After 18 h,the DMSO was evaporated (50° C., 0.1 torr) and the residue wastriturated with acetone/ether (20/70, 2×50 mL) and then acetone (2×50mL). The residue was dried on high vacuum overnight to give an orangesolid (orange solid, 2.00 g) which was carried on to the next stepwithout further purification.

Trifluoroacetic acid (TFA, 5.36 mL) was added dropwise to a suspensionof crude 3 (1.09 g, 1.34 mmol) in CH₂Cl₂/H₂O (5/1, 6 mL). After gentlyshaking for 2 h, the mixture was concentrated in vacuo and the residuewas azeotropically dried with dry toluene (10 mL). The residue wastriturated with CH₂Cl₂ (5×5 mL); the yellowish CH₂Cl₂ supernatant wasremoved each time with a pipette and gradually became less yellow witheach wash until nearly colorless. The crude 4 (thick red oil) was usedin the next step without further purification.

DIPEA (1.13 mL, 6.50 mmol, 5.00 equiv) was added dropwise to asuspension of crude 4 (0.891 g, 1.30 mmol) in anhydrous DMSO (6 mL).After 15 min, a solution of NHS EET analog ester 5 (0.569 g, 1.30 mmol)in dry CH₂Cl₂ (6 mL) was added. After 24 h, the reaction mixture wasdiluted with ice-cold 20% acetone/Et₂O (50 mL). The supernatant wasdecanted away from the resulting precipitate and the residue wastriturated sequentially with additional 20% acetone/Et₂O (50 mL), 50%acetone/Et₂O (100 mL), 0.1 N HCl (20 mL), and then acetone (2×50 mL).The residue was dried under high vacuum overnight to give EET-F01 (6) asan orange solid (0.690 g, 59%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.65; (s, 1H), 7.93-7.80; (m, 1H),7.73-7.58; (m, 2H), 6.95; (s, 1H), 6.64; (dd, J=8.7, 2.3 Hz, 2H),5.79-5.69; (m, 2H), 5.38-5.28; (m, 2H), 4.49; (d, J=6.1 Hz, 2H),3.52-3.45; (m, 4H), 3.22-3.13; (m, 4H), 3.01-2.87; (m, 4H), 2.10-2.09;(m, 2H), 2.05; (t, J=7.5 Hz, 2H), 2.01-1.93; (m, 4H), 1.51-1.40; (m,2H), 1.40-1.14; (m, 18H), 0.86; (t, J=7.0 Hz, 3H). LCMS (ES-APCI⁺)Calcd. for [C₄₄H₆₇N₁₁O₉]^(+ 894.1,) Found 894.4.

Synthesis of NHS EET analog ester 5. N-Hydroxysuccinimide (NHS, 0.165 g,1.43 mmol, 1.10 equiv) and dicyclohexylcarbodiimide (DCC, 0.322 g, 1.56mmol, 1.20 equiv) were added to a stirring 0° C. solution of EET analog³7 (0.443 g, 1.30 mmol) in dry EtOAc (26 mL). After 5 min,N,N-dimethylpyridine (DMAP, 15.9 mg, 0.13 mmol, 0.100 equiv) was addedand the suspension was warmed to rt. After 24 h, the reaction wasfiltered through a pad of Celite™ and the Celite™ bed was washed withEtOAc. The filtrate was washed with saturated aq. NaHCO3 (10 mL) andbrine (10 mL). The organic layer was dried over Na2SO4, filtered, andconcentrated in vacuo to give crude 5 which was used in the next stepwithout further purification.

Synthesis of EET-A Folate Conjugate:

Synthesis of 9. A mixture of EET-A disodium salt³ (100 mg, 0.20 mmol),N-(3 -dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI.HCl,88 mg, 0.460 mmol, 2.3 equiv), and hydroxybenzotriazole (HOBt, 6.10 mg,0.04 mmol, 0.2 equiv) in anhydrous DMF (1 mL) was stirred at rt. After 2h, 2 (188 mg, 0.481 mmol, 2.40 equiv) in dry CH₂Cl₂ (1 mL) and DIPEA (79μL, 0.440 mmol, 2.20 equiv) were added sequentiall. Following anadditional 40 h, the mixture was diluted with MeOH (10 mL) andconcentrated in vacuo. The crude product was purified using a TeledyneIsco Combiflash® R_(f) chromatographic system (4 g SiO2 column) elutedwith a gradient of 0-100% EtOAc/hexane over 25 min; 100% EtOAc for 15min; and 10% MeOH/CH₂Cl₂ for 7 min to give 9 (169 mg, 70%).

¹H NMR (500 MHz, CD₃OD) δ 7.51-7.44; (m, 14H), 7.33-7.25; (m, 14H),7.24-7.17; (m, 6H), 5.87; (s, 1H), 5.40-5.33; (m, 2H), 4.69; (dd, J=7.5,5.9 Hz, 1H), 3.71-3.49; (m, 18H), 3.17-3.07; (m, 4H), 2.62; (dd, J=15.0,6.0 Hz, 1H), 2.53; (dd, J=15.0, 7.7 Hz, 1H), 2.35; (t, J=5.5 Hz, 4H),2.21; (t, J=7.6 Hz, 3H), 2.13-1.99; (m, 4H), 1.66-1.54; (m, 2H),1.53-1.45; (m, 4H), 1.43-1.26; (m, 14H), 0.93; (t, J=6.9 Hz, 3H).

Synthesis of 10. Trifluoroacetic acid (TFA, 0.40 mL) was added dropwiseto a mixture of trityl-protected amine 9 (130 mg, 0.108 mmol) in H₂O(0.50 mL) and CHCl₃ (1.5 mL). After 45 min, the reaction was dilutedwith MeOH (3 mL) and concentrated in vacuo. Co-evaporation with toluene(3×5 mL) gave 10 as a white solid which was taken to the next stepwithout further purification.

Synthesis of 11. DIPEA (77 μL, 0.432 mmol, 4 equiv) was added to amixture of crude 1 (116 mg, 0,216 mmol, 2 equiv) and 10 (77 mg, 0.108mmol) in anhydrous DMSO (1.50 mL). After 40 h, the mixture was pouredwith stirring into 0° C. Et₂O (25 mL). The supernatant was carefullydecanted from the resulting precipitate and the precipitate wastriturated with acetone/Et₂O (30/70, 30 mL) and acetone/CH₂Cl₂ (30/70,30 mL). The residue was dried under high vacuum to give 11 (57 mg) as anorange solid.

¹H NMR (500 MHz, DMSO-d₆) δ 8.73-8.61; (m, 2H), 8.12-7.83; (m, 4H),7.76-7.55; (m, 3H), 7.03-6.88; (m, 2H), 6.64; (dd, J=7.8, 4.5 Hz, 4H),5.76; (br s, 2H), 5.40-5.20; (m, 2H), 4.57-4.42; (m, 5H), 3.68-3.44; (m,14H), 3.22-3.08; (m, 6H), 3.06-2.88; (m, 6H), 2.02-1.84; (m, 7H),1.53-1.14; (m, 18H), 0.92-0.78; (m, 3H).

The kidney-targeted EET analogs described herein may be formulated witha pharmaceutically acceptable carrier or excipient to form a primarytherapeutic agent. “A pharmaceutically acceptable carrier” is a materialthat can be used as a vehicle for administering a therapeutic orprophylactic agent, (e.g., kidney-targeted EET analog), because thematerial is inert or otherwise medically acceptable, as well ascompatible with the agent.

Also disclosed are kits that include the conjugates and/or complexestogether with a pharmaceutically acceptable excipient to form atherapeutic agent. The kit may include an implement for administeringthe therapeutic agent. In addition, the kit may include one or moresupplemental therapeutic agents and/or diagnostic agents.

Advantageous properties of kidney targeted EET analogs as describedherein also include: targeted delivery of EET analogs to the kidneys,therefore leveraging the useful biological properties of EETs (e.g.,vasodilation, anti-inflammation, anti-apoptosis, and anti-fibrosis) in atargeted manner; utility as a prophylactic to prevent or limitnephrotoxicity as well as an interventional therapy; effective atten-fold lower doses than untargeted EET counterparts; and fewersystemic side effects.

In another aspect, provided herein are methods of treating kidneydisease in a subject in need thereof with a compound as describedherein. Also provided herein are methods for delivering a therapeuticagent to a target cell population comprising a folate receptor, themethod comprising providing the compound of claim 1 and contacting thetarget cell population with an effective amount of the compound topermit binding of the compound to the folate receptor.

Therapeutic and preventative applications of the kidney-targeted EETanalogs described herein include, without limitation, treatment ofkidney diseases that begin at the proximal tubules, acute tubularnecrosis (ATN), and ischemic renal injury following bypass or transplantsurgery; treating or preventing drug-induced nephrotoxicity, includingCisplatin-induced nephrotoxicity; use as a preservative to store kidneysto be transplanted; treatment of diabetic nephropathy, Focal SegmentalGlomerulosclerosis (FSGS), and Chronic Kidney Disease (CKD). Othertherapeutic and prophylactic applications of the kidney targeted EETanalogs provided herein include treatment of and/or protection fromdrug- and radiation-induced kidney damage, hypertension and cardiorenalsyndrome kidney damage, and metabolic syndrome and diabetes nephropathy.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, preferred methodsand materials are described herein.

The following Example is illustrative of certain embodiments of theinvention. The examples, methods, and conditions presented therein arenot to be construed as limiting the scope nor the spirit of theinvention.

EXAMPLES Example 1—Kidney-Targeted Delivery of EET Analogs

Epoxyeicosatrienoic acid (EET) analogs have exceptional therapeuticpotential to combat cardiovascular and kidney diseases. EET analogscombat damage in acute and chronic kidney disease models. Biologicalactions attributed to EET analogs such as vasodilation,anti-inflammation, anti-apoptosis, and anti-fibrosis are ideally suitedto treat kidney diseases. Although EET analogs have performed well inseveral in vivo models, targeted delivery of EET analogs to the kidneycan be reasonably expected to reduce the level of drug needed to achievea therapeutic effect in the kidney and obviate possible side effects.

For EET analog kidney-targeted delivery, we conjugated an EET analog tofolic acid because there is a high concentration of folate receptors inrenal tissue. The EET analog was conjugated to folic acid via aPEG-diamine linker. Next, we compared the kidney targeted EET analog,EET-FO1, to a well-studied EET analog, EET-A. EET-A or EET-FO1 wasinfused i.v. (10 mg/kg/hr) for 6 hours via the rat jugular vein. Plasmaand kidney tissue were collected and EET-A or EET-FO1 measured byLC-MS-MS. EET-A plasma level was 1.6 ng/mL, but EET-A was undetectablein the kidney. On the other hand, EET-FO1 was 6.5 ng/mL in plasma and26.7 ng/mL in kidney tissue. These data demonstrate that EET-FO1 targetsthe kidney. Experiments were conducted to compare EET-FO1 and EET-A todecrease cisplatin-induced nephrotoxicity. A single injection ofcisplatin (7 mg/kg ip) was administered to WKY rats treated withvehicle, EET-A (10 mg/kg ip) or EET-FO1 (20 mg/kg or 2 mg/kg ip) forfive days. Cisplatin increased BUN (125±11 mg/dL) and NAG (12±4 IU/L)compared to control (36±9 mg/dL and 4±1 IU/L). EET-FO1 was as effectiveas EET-A in decreasing BUN, NAG, and renal histological injury five daysfollowing cisplatin administration. Despite it almost 2×-greatermolecular weight compared with EET-A, EET-FO1 was effective in loweringBUN and NAG at 20 mg/kg/d and at a 10-fold lower dose of 2 mg/kg/d.These data clearly demonstrate that EET-FO1 targets the kidney andallows for a lower effective dose. In conclusion, we have developed akidney targeted EET analog, EET-FO1 that demonstrates excellentpotential as a therapeutic for kidney diseases.

Example 2—Protection Against Cisplatin-Induced Nephrotoxicity UsingKidney-Targeted EET Analogs

Cisplatin is a platinum-based inorganic chemotherapy agents available totreat a variety of malignancies (1-3). However, several adverse sideeffects are associated with the use of cisplatin in clinical practice.Among several, the most common adverse effect is severe nephrotoxicityoccurs in 25-40% of cisplatin treated patients that affects theeffective use of this chemotherapeutic agent (4-7). It is widelyacknowledged that there is a need to develop agent that could protectthe kidney from the adverse effect of cisplatin during its clinical use.One approach that can be used to develop kidney protective agent againstcisplatin nephrotoxicity is targeting CYP epoxygenase pathway whichproduce epoxyeicosatrienoic acids (EETs) from arachidonic acids.

Several studies demonstrated that EETs display myriad of biologicalactions useful for organ protection, including the kidney (8,9). Inthese studies, we have demonstrated that synthetic EET mimetics kidneyprotective in numerous pathological conditions like hypertension,cisplatin nephrotoxicity and radiation nephropathy (10-14). Thesestudies clearly demonstrated potential of EET-based approach to protectkidney in pathological situations of different etiologies and we havedeveloped several synthetic EET analogs that have EET mimetic property.

Recently, attempts have been made to develop EET analogs that possessEET-mimetic activity along with a unique property to target the kidney.We have developed a novel form of kidney targeted EET analog byconjugating EET pharmacophore with folic acid. In preliminary studies,we investigated its ability to target kidney in compare to the EETanalog without any folic acid conjugation. We also compared the abilityof the folic acid conjugated EET analog with the original EET analogwithout any folic acid moiety in protecting the kidney against cisplatinnephrotoxicity.

MATERIALS AND METHODS

All chemicals and assay kits were purchased from Sigma Aldrich (St.Louis, Mo., USA) unless and otherwise mentioned. EET analogs weredesigned and synthesized in the laboratory of John R. Falck, Departmentof Biochemistry, University of Texas Southwestern Medical Centre,Dallas, Tex.

Animal studies: All animal studies were approved and carried outaccording to guidelines of the Institutional Animal Care and UseCommittee, Medical College of Wisconsin. Animals were kept in atemperature-controlled environment with a 12-h light/dark cycle and wereallowed free access to food and water. An acclimatization period of 6days was allowed for the rats before experimentation.

Acute study: Overnight fasted (12-14 hrs) male SD rats (10 weeks old)were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). After atracheostomy, the left jugular vein was cannulated to continuouslyinfuse vehicle or EET-A or folate conjugated EET analog at a rate of 6ml/kg/hr for 6 hours. Plasma and kidney sample were collected at the endof the protocol, snap-frozen in liquid nitrogen and stored at −80° C.until analyzed for LC-MS. EET-A and folate conjugated EET analogsolutions were prepared in 0.1% DMSO and 1% PEG-400 in saline.

Chronic study: In chronic study, male Wistar-Kyoto (WKY) rats weighing180-200 g (Charles River, Mass., USA) were used. The rats were assignedinto four groups (n=4 in each group). Rats of group 1 received drinkingwater ad libitum and on day 7 administered DMSO (300-500 μl i.p.), whichis used to prepare cisplatin solution. While, rats of groups 2,3,4&5were pre-treated with vehicle (20% DMSO in PEG-400 v/v), EET analog (EETanalog in double distilled water) or novel folate conjugated EET analogF01 at 20 mg/kg and 2 mg/kg doses (prepared in a mixture of 20% DMSO inPEG400), respectively in osmotic mini pump (2ML, Alzet, Cupertino,Calif.) for seven days. On day 7 these rats were administered with asingle dose of cisplatin (7 mg/kg i.p.) followed by five-days treatmentvehicle, EET-A and F01. One day before euthanasia, the urine of each ratwas collected over a 24-h period. Five days after cisplatin or vehicleadministration, rats were anesthetized for blood sample collectionfollowed by euthanasia. A schematic of the protocol is depicted in FIG.1.

Biochemical analysis: The levels of blood urea nitrogen (BUN) (BioAssaySystems, Hayward, CA, USA) and the activity of urinaryN-acetyl-β-(D)-glucosaminidase (NAG) in the urine was measured by a kitfrom Diazyme (Diazyme Laboratories, Poway, Calif., USA).

Mass spectrometric analysis: Plasma and kidney levels of EET-A andfolate conjugated EET-A were measured by LC-ESI-MS method. Samples wereprepared from 200 μl of plasma or kidney homogenate from EET-A or folateconjugated EET-A treated rats using solid phase extraction with VarianBond Elut® C18 column (Agilent Technologies, Santa Clara, Calif., USA).The extracted samples were stored at −80° C. before analysis. Sampleswere warmed to room temperature, dried in a stream of nitrogen and theresidue reconstituted in 20 μL of acetonitrile, 12 μL injected.Components were resolved on a 250 mm×2.0 mm Kromasil C18-column packedwith 5 μm diameter particles having 100 Å pores. Gradient elution from80% A to 10% A was used with elutant flow of 0.2 mL/min. Solvent A waswater with 0.01% formic acid and solvent B was acetonitrile with 0.01%formic acid using the following profile: 20% B to 30% B in 10 min, 30% Bto 60% B in 17 min, 60% B to 90% B in 28 min, hold at 100% for 7 min,then 7 min re-equilibration. MS/MS analysis was performed on an Agilent6460 triple quadrupole mass spectrometer equipped with a Jet Stream™interface. Precursor ion, product ion, collision energy and fragmentervoltage were optimized for each compound in negative polarity. Otherparameters were as follows: drying gas flow=10 L/min at 325° C.,nebulizer=20 psi, sheath gas flow=11 L/min at 325° C., capillary=3.5 kV,and nozzle=1.0 kV. Results acquired at unit-mass resolution.

RESULTS

Plasma and Kidney Content of F01 is Higher than EET-A after AcuteIntravenous Administration

In the acute animal study, equal doses of EET-A and F01 wereadministered intravenously for 6 hours followed by plasma and kidneytissue collection. We demonstrated 5-fold higher plasma level of F01compared to EET-A after 6-hour continuous administration of F01 andEET-A (FIG. 2). Interestingly, we also demonstrated that after 6-hourcontinuous administration the kidney content of EET-A was almostun-detectable compared to F01 content in the kidney (FIG. 2).

Chronic Treatment of F01 Demonstrated Better Kidney Protective Actionthan EET-A Against Cisplatin Nephrotoxicity

In a separate set of experiment groups of rats were pre-treated with F01and EET-A before induction of cisplatin nephropathy and then furthertreated with the test compounds (F01 and EET-A) for another 5 days. Thekidney protective actions of F01 and EET-A is determined from the levelof blood urea nitrogen (BUN) and urinary content of NAG. We demonstrateda better kidney protective action of F01 compared to EET-A, thenon-folate form of EET analog (FIG. 3).

CONCLUSION

The folate conjugated form of an EET analog, F01 has ability to reachthe plasma and the kidney in a faster rate than its non-folateconjugated form. Also, the F01 compound demonstrated superior kidneyprotective action than EET-A against cisplatin nephrotoxicity.

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The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

1. A compound comprising an EET analog conjugated to folic acid ormethotrexate, wherein the EET analog used to prepare the compound has aformula selected from the group consisting of


2. The compound of claim 1, wherein the EET analog is conjugated tofolic acid.
 3. The compound of claim 1, wherein the EET analog is


4. The compound of claim 1, wherein the EET analog is


5. The compound of claim 1, wherein the EET analog is


6. The compound of claim 1, wherein the EET analog is


7. A pharmaceutical composition comprising the compound according toclaim 1 and at least one component selected from the group consisting ofa pharmaceutically acceptable carrier, excipient, or diluent.
 8. Amethod of treating kidney disease in a subject in need thereof, themethod comprising administering a therapeutically effective amount ofthe pharmaceutical composition of claim 7 to the subject, whereby thekidney disease is treated.
 9. A method of reducing nephrotoxicity in asubject in need thereof, the method comprising administering atherapeutically effective amount of the pharmaceutical composition ofclaim 7 to the subject, whereby the nephrotoxicity is reduced.
 10. Themethod of claim 9, wherein the nephrotoxicity is drug-induced.
 11. Themethod of claim 9, wherein the nephrotoxicity is cisplatin-induced. 12.A pharmaceutical composition comprising the compound according to claim2 and at least one component selected from the group consisting of apharmaceutically acceptable carrier, excipient, or diluent
 13. A methodfor delivering a therapeutic agent to a target cell populationcomprising a folate receptor, the method comprising providing thepharmaceutical composition of claim 12 and contacting the target cellpopulation with a therapeutically effective amount of the pharmaceuticalcomposition to permit binding of the compound to the folate receptor.